All vertebrate hemoglobins have the same molecular configuration. Hemoglobin is a protein made up of four polypeptide subunits, two alpha chains and two non-alpha chains. A natural cavity is defined by certain amino acids in each subunit. This cavity contains a heme prosthetic group, consisting of a porphyrin ring and an iron ion. Hemoglobin in the form capable of reversibly binding and releasing oxygen, the iron ion is in the +2 oxidation state, i.e., the ferrous form, and is sequestered by each porphyrin ring within the protein. In a hemoglobin tetramer, each alpha subunit is associated with a non-alpha subunit to form two stable alpha/non-alpha dimers, which in turn associate to form the tetramer. The subunits are noncovalently associated through Van der Waal's forces, hydrogen bonds and salt bridges. The molecular weight of native human hemoglobin is about 65,000 Daltons.
Hemoglobin can be collected from mammalian blood or derived from genetically engineered sources. However, even after stringent purification, unmodified vertebrate hemoglobin has no therapeutic utility. Free hemoglobin has an affinity for oxygen too high for release of oxygen to the tissues. Further, unmodified free vertebrate hemoglobin readily dissociates into alpha/non-alpha dimers in the circulation. High concentrations of these dimers overwhelm the haptoglobin scavenging system and accumulate in the tubules of the kidney, where they are nephrotoxic. Chemical modification of hemoglobin is necessary to overcome these deficiencies.
The tetrameric structure of hemoglobin may be stabilized by intramolecular covalent crosslinking between at least two of the subunits of the native hemoglobin. The molecular weight of the resulting hemoglobin composition is about 65,000 Daltons, similar to that of the source hemoglobin. Moreover, the manner of intramolecular crosslinking may be selected to provide both stabilization of the tetrameric structure of hemoglobin and a change in hemoglobin conformation sufficient to impart oxygen binding characteristics similar to those in freshly collected red blood cells. For example, native hemoglobin may be extracted from red blood cells, purified, and intramolecularly crosslinked. Examples of crosslinked hemoglobins and methods for their preparation are described in U.S. Pat. Nos. 4,001,401 and 4,053,590, which disclose intramolecular crosslinking between an alpha and beta subunit of a hemoglobin tetramer utilizing compounds such as halogenated cycloalkanes, diepoxides, and diazobenzidines. WO 90/13309 (Staat der Nederlanden de Minister Van Defeuric) discloses a method for crosslinking hemoglobin through a beta-beta subunit linkage. In the present method, a preferred modified hemoglobin is crosslinked with bis(3,5-dibromosalicyl) fumarate to create a fumarate crosslink between the two alpha subunits. This crosslinked hemoglobin is more fully described, together with methods for its preparation, in U.S. Pat. Nos. 4,598,064, 4,600,531, RE 34,271, omitting the chromatography step. It is preferably manufactured under the conditions disclosed in U.S. Pat. No. 5,128,452 (Hai) to prevent crosslinking between the beta chains. The preferred diaspirin crosslinked hemoglobin will hereafter be referred to as "DCLHb". U.S. Pat. Nos. 4,598,064, 4,600,531, RE 34,271, and 5,128,452 are hereby incorporated by reference. In addition, the genes encoding subunits of a desired naturally occurring or mutant hemoglobin can be cloned, placed in a suitable expression vector and inserted into an organism, animal, or plant, or into cultured animal or plant calls or tissues. The hemoglobin produced therefrom can be expressed and collected as described, for example, in Hoffman, S. J. and Nagai, K. in U.S. Pat. No. 5,028,588. Transgenic animals can be produced that express non-endogenous hemoglobin (Logan, J. S. et al., PCT application WO 92/22646).
These intramolecularly crosslinked hemoglobin compositions have therapeutic utility both for humans and other mammals. See, for example, Sloan, Koenigsberg, and Bickell, "The Use of Diaspirin Cross-linked Hemoglobin (DCLHb) in the Hospital Management of Hemorrhagic Hypovolemic Shock", Academic Emerg Med 1995; 2(5): Abstract No. 78.
Hemoglobin compositions having a molecular weight of about 65,000 Daltons have a short half-life in the circulatory system, because they are able to traverse cellular pores in the membrane of the blood vessels and capillaries, entering the interstitial spaces between endothelial cells lining the lumen. Consequently, these hemoglobin compositions are lost from the circulation while they still retain therapeutic utility. The hemoglobin composition diaspirin crosslinked hemoglobin (DCLHb), which has a molecular weight of about 65,000 Daltons, has an elimination half-life of 2.5 hours for a 25- and 50-mg/kg dose and an elimination half-life of 3.3 hours for a 100 mg/kg dose. (Przybelski, et al., Crit Care Med 1996; 24(No. 12): 1993-2000.)
Further chemical modifications to hemoglobin which increase molecular weight have been used in attempts to extend the duration of circulation of the hemoglobin composition. See Bunn, H. F., Amer J Hematol 42:112-117, 1993. These additional modifications include conjugation and polymerization. In addition, modification to increase overall negative charge may also extend half-life in the circulation, since the negative charges in the vessel walls tends to repel hemoglobin with high negative charge.
Conjugated hemoglobin is hemoglobin to which a non-protein macromolecule is bound covalently. The properties of hemoglobins conjugated to polysaccharides have been reviewed by Dellacherie. (E. Dellacherie, "Polysaccharides in Oxygen-carrier Blood Substitutes", chapter 17 in Polysaccharides in Medicinal Applications, S. Dumitriu, ed. Marcel Dekker, Inc., New York, 1996, pages 525-545.) For example, hemoglobin may be conjugated to inulin in a process disclosed in U.S. Pat. No. 4,377,512 (Ajinomoto). Hemoglobin may be conjugated to a polysaccharide such as dextran in a process disclosed in U.S. Pat. No. 4,900,816 (Fisons). Macromolecular conjugates of hemoglobin and a substituted dextran, together with a process for its preparation, are provided in U.S. Pat. Nos. 5,079,337 and 5,110,909 (Merieux). A further example of a conjugated hemoglobin composition is a hemoglobin chemically modified by polyalkylene glycol, which is described together with a process for its preparation in WO 91/07190 (Enzon). An example of a hemoglobin conjugated to poly(alkylene oxide) and a process for its preparation are provided in U.S. Pat. Nos. 4,301,144, 4,412,989, and 4,670,417, and in Japanese Patent Nos. 59-104323 and 61-053223 (Ajinomoto).
A polymerized hemoglobin is one in which intermolecular crosslinking of hemoglobin tetrameres has been used to increase the molecular weight of the modified hemoglobin. An example of a polymerized hemoglobin and a process for its preparation are described in U.S. Pat. No. 4,777,244 which discloses a method for crosslinking and polymerizing with aliphatic dialdehydes.
A hemoglobin, modified by a combination of methods, is exemplified by the following. Hemoglobins modified by pyridoxal-5'-phosphate to adjust the oxygen affinity and by polyethylene glycol conjugation and processes for its preparation are described in Japanese Patent Nos. 59-089629, 59-103322 and 59-104323 (Ajinomoto). U.S. Pat. No. 5,248,766 discloses a crosslinking polymerizing strategy and a process for covalently interconnecting intramolecularly crosslinked tetrameric units with oxiranes to form polyhemoglobins with molecular weights in excess of 120,000 Daltons.
Even though conjugation and polymerization both increase the molecular weight of the constituent hemoglobin, these hemoglobin do not have isotropically increased size. This is illustrated in the ball and stick structures of FIG. 1. The process of polymerization with a bifunctional reagent such as glutaraldehyde generates a homologous series of hemoglobin polymers having, for the most part, linear structures analogous to barbells or beads on a string FIGS. 1a and 1b. Since the polymers comprise multiple hemoglobin units, the molecular weight of each of the component polymers is about (64,500).times.n, where n has a value from 2 to 10 or more. If one considers the functional diameter of the polymeric hemoglobin stretched lengthwise along an arbitrary x-axis, it is larger than that of a single hemoglobin. However, if one considers the functional diameter of the polymeric hemoglobin from the perspective of either terminus (i.e., along the z-axis) as in FIG. 1c), this diameter is no greater than that of a single hemoglobin molecule. Therefore, the polymeric hemoglobins can still readily leak out of capillaries through the pores of the luminal membranes and interstitial junctions, thereby shortening the period of therapeutic effectiveness. Similarly, conjugation with a polysaccharide such as dextran or inulin generates a conjugated hemoglobin having a linear structure analogous to the barbell structure in FIG. 1a. Thus, conjugates of this type also are characterized by a functional diameter no greater than that of a single hemoglobin molecule.
Other problems arise in conventionally conjugated hemoglobin. Conjugation of hemoglobin with a polyalkylene oxide, such as polyethylene glycol (PEG) or polyoxyethylene, or polymerization of hemoglobin undesirably increases its viscosity (Winslow, R. M., "The Design of Blood Substitute Oxygen Carriers for Clinical Applications", In: Shock: From Molecular and Cellular Level to Whole Body, K. Okada et al., editors. Elsevier Science, 1996, pages 323-333). Viscous fluids are difficult to administer, and would unacceptably alter the fluid properties of the blood.
A hemoglobin uniformly greater in diameter than the diameters of the cellular pores would be retained for longer periods of time in the circulatory system, particularly if the surface modification also isotropically dispersed negative surface charge would be repulsed from the surface of endothelial cells lining the lumen of the circulatory system. See Rennke, Cotran, Venkatachalam, J Cell Biol 67:638, 1975. No known hemoglobin composition meets these criteria.